(a) Field of the Invention
This invention relates to a method and device for detecting pregnancy. More specifically, this invention concerns a simple and sensitive method and device for the detection of pregnancy in women; the method and device being especially useful for the detection of pregnancy in its very early stages.
(B) Prior Art
A simple, sensitive test for the early diagnosis of human pregnancy would be an important contribution to medicine and society. For instance, it would be advantageous in cases of unwanted pregnancies or in cases of habitual aborters who would benefit from early therapy. It would also be advantageous for the physician to have knowledge of an early pregnancy before prescribing a drug that may be teratogenic, or in those instances where a woman has unwittingly been exposed to a possibly teratogenic drug. Also, not to be overlooked, is the all important psychological factor for the woman to know for certain whether she is pregnant or not.
The most widely used pregnancy tests employed today are those based on the detection of human chorionic gonadotropin (HCG) in urine samples by immunological methods. These tests rely on the fact that HCG is the gonadotropin of pregnancy, being secreted by the chorionic tissue of the placenta in increasing amounts soon after the implantation therein of a fertilized ovum. [The peak secretion of HCG of more than 50,000 i.u. per a 24 hour collection of urine occurs between 56 and 84 days after the last menstrual period, E. H. Venning in "Text Book of Gynecologic Endocrinology", J. J. Gold, Ed., Harper and Row, New York, 1968, pp. 95-97]. These tests are generally reliable for detecting pregnancy after about the twelfth day following a missed menstrual period (i.e., about the fortieth day of amenorrhea) giving about a 2 to 6% error when correctly performed, B. M. Hibbard, Brit. Med. J., 1, 593 (1971) and C. A. Horwitz, et al., Obstet. Gynecol., 43, 693 (1974). However, the tests cannot be relied upon prior to that time since they only can detect minimum concentrations of HCG of about 1000-3000 m. i.u./ml of urine. The main reason for not increasing the sensitivity of these test by concentrating urine samples is to avoid false positives resulting from substances which cross react with the HCG-antiserum, B. M. Hobson, J. Reprod. Fertil., 12, 33 (1966).
Recently, more sensitive tests have been developed. These newer tests are based on sensitive but sometimes non-specific radioimmunoassay (RIA) techniques. The non-specificity of these tests arise from the fact that they also give positive results with other gonadotropins such as human pituitary luteinizing hormone (LH), i.e., the antisera to HCG may cross react with LH. Using the non-specific RIA techniques, LH-HCG has been shown to rise sharply beginning 10 to 14 days after the mid-cycle LH peak in the first month of pregnancy. For example, see R. B. Jaffe, et al., J. Clin. Endocrinol. Metabol., 29, 1281 (1969); A. F. Parlow, et al., J. Clin. Endocrinol. Metabol., 31, 213 (1970); D. P. Goldstein, et al., Fertil. Steril., 23, 817 (1972); L. Wide, Lancet, 2, 863 (1969) and D. R. Mishell, Jr., et al., Am. J. Obstet. Gynecol., 117, 631 (1973).
A RIA also has been developed which is specific for the beta subunit of HCG. This latter test has been used to measure serum or plasma HCG by RIA in the presence of circulating LH during the same early period of pregnancy. See, for example, T. S. Kosasa, et al., J. Clin. Endocrinol. Metabol., 36, 622 (1973) and T. S. Kosasa, et al., Obstet. Gynecol., 42, 868 (1973).
Although RIA techniques are sensitive, it will be appreciated that these methods are expensive and complex. They must be performed by highly trained personnel using isotopic material and very sophisticated equipment.
Another test is the radio-receptor assay for HCG recently developed by B. B. Saxena, et al., Science, 184, 793 (1974). Although less time-consuming than the RIA, this test also involves radioactivity, special equipment and an operator with technical skills.
Another group of tests for pregnancy are the biological tests, including the well known "rabbit test". For a review on these and other tests see B. M. Hobson, cited above. It is a well known fact, however, that these biological tests are laborious and time-consuming. Furthermore, they require the maintenance of colonies of animals which are subjected to seasonal variations in sensitivity.
Still other pregnancy tests have been reported. These other tests depend on estimations of serum steroid levels or on the observation of withdrawal bleeding after progestogen alone or progestin-estrogen therapy. These tests are considered to be less reliable, and in the case of the latter, unsafe to the subject and conceptus.
In accordance with the need for a simple, sensitive, reliable and safe test for the detection of pregnancy, the present invention provides a method and a device for such a test based on the concept of ultrafiltration of body fluid (e.g., urine, serum or plasma) followed by immunological determination of HCG. Ultrafiltration had been used to concentrate initially high titres of "trophoblastic tumor HCG", in urine. M. L. Taymor, et al., J. Endocrinol., 36, 417 (1966) and S. Lok, Asian J. Med., 9, 319 (1973). Such tumors produce high levels of "trophoblastic HCG", much higher than those encountered in pregnancy. Taymor, et al. concentrated the high "trophoblastic titre HCG" urine in a step directed to the purification of this gonadotropin. Incidentally, these tumors occur only rarely. It should be noted that samples from subjects having trophoblastic tumors will give a positive test in the present invention. Accordingly, in the case where the present method gives a positive test which is later shown to be false in regards to pregnancy, such HCG producing tumors should be suspected.
It is not established whether the chorionic gonadotropins of pregnancy have carbohydrate and protein structures that are identical to the chorionic gonadotropins secreted by patients with hydatiform male or metastatic carcinoma, R. E. Canfield, et al., Recent Progr. Hor. Res, 27, 121 (1971). Several reports have suggested that the urine from patients with choriocarcinoma may contain a gonadotropin that is chemically or immunologically different from the HCG of pregnancy, R. A. Reisfeld and R. Hertz, Biochem. Biophys. Acta, 43, 540 (1960); E. E. Wilde and K. D. Bagshawe, Ciba Found. Study Group [Pap,] 22, 46 (1965) and N. Mochizuki, et al., Folia Endocrinol. Jap., 44, 459 (1968).
The present invention provides a convenient method and device for detecting pregnancy, especially in the early stages, the method being reliable and easily performed. Furthermore, the invention provides a method giving a substantial reduction in false negatives and false positives compared to prior art non-radioactive methods.
U.S. Pat. No. 4,033,723 issued July 5, 1977, of Givner and Schilling, herein incorporated by reference, discloses a method and device suitable for the detection of pregnancy, including early pregnancy, based on the ultrafiltration of the biological fluid, selected from the group of urine and serum containing human chorionic gonadotropin of the .beta.-subunit thereof and detection by immunological means of the concentrate, is provided. The device includes a chamber, opened at the top and closed at the bottom, having the upper portion of at least one wall formed of an ultrafiltration membrane permeable to urine or serum and capable of selective retention of human chorionic gonadotropin, all other walls being formed of a rigid impermeable material, and a layer of absorbent capable of sorbing urine or serum passing through the membrane, the absorbent being contiguous to the outside surface of the membrane and in effective contact with the membrane. The chamber further includes a lower portion, all walls of which are impermeable, for retaining a fixed volume of urine, serum concentrate containing human chorionic gonadotropin, outlet means in the lower portion of the chamber, means for opening the outlet means, and a reagent receptacle connected to the chamber through the outlet means, the reagent receptacle adapted to receive a reagent for the immunological determination of human chorionic gonadotropin or its .beta.-subunit and means for viewing the reaction of the reagent therein with the said urine or serum concentrate containing human chorionic gonadotropin.
The instant invention comprises carrying out the above described method or treating the above described device such that the concentration by ultrafiltration is conducted in the presence of a particular fraction of polyvinylpyrrolidone, and using a particular immunological reagent.
Thus, one embodiment of the invention resides in a pregnancy test method suitable for the detection of the human chorionic gonadotropin of pregnancy in urine at a concentration as low as about 40 m.I.U./ml of urine and capable of detecting pregnancy as early as day 26 of a regular 28 day menstrual cycle. The test method includes the steps of:
(a) clarifying a sample of the urine to be tested; PA1 (b) subjecting about 5 to 50 ml of the clarified urine sample to absorbent induced ultrafiltration through an ultrafiltration membrane backed by a layer of absorbent capable of sorbing urine serum passing through the membrane, and said membrane having a molecular weight cut-off from about 15,000 to about 35,000, whereby human chorionic gonadotropin is retained in the sample; PA1 (c) continuing the ultrafiltration until the retentate sample is one-tenth to one-five hundredth its original volume; PA1 (d) diluting the retentate with sufficient water to provide a retentate sample of about 0.5 ml; and PA1 (e) contacting the retentate sample with the appropriate amount of an immunologic reagent for detecting the presence of human chorionic gonadotropin in the retentate sample. PA1 (1) pyruvic aldehyde stabilized erythrocytes sensitized to human chorionic gonadotropin with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinialdehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethylmalonimidate dihydrochloride, dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; and PA1 .eta. rel= viscosity of solution compared with solvent
The invention resides in the improvement which comprises pre-washing said membrane with a dilute solution of polyvinylpyrrolidone prior to or concurrent with ultrafiltration, wherein said polyvinylpyrrolidone has an average molecular weight greater than about 9,000 and less than about 40,000; particularly wherein the immunologic reagent includes:
(2) a highly purified antiserum to human chorionic gonadotropin or the beta subunit thereof.